Project Summary Trans?synaptic bidirectional tracing tools for imaging and omics analysis A central question in systems neuroscience is how hormones, such as estrogen, regulate animal behavior at the level of synapses and circuits. The Shah lab has recently identified hormone-responsive neuronal populations in the hypothalamus and amygdala that mediate distinct behaviors between the sexes. We and others are keenly interested in identifying the post-synaptic targets of these neurons, so that we can discover the mechanisms by which these neurons guide distinct behaviors that are exhibited by the two sexes, such as during mating. However, existing tools for discovering synaptically connected neurons ? such as rabies virus-based tracers ? work only in the retrograde direction, from post-synapse to pre- synapse, and can be toxic, giving rise to artifacts. We and the larger community of systems neuroscientists are urgently in need of new technologies for accurate, non-toxic trans-synaptic tracing in both retrograde and especially anterograde (from pre- to post-synaptic) directions, so that we can discover the upstream and downstream targets of functionally important neuronal populations. To address this unmet need, the Shah lab proposes to collaborate with the Ting lab, who are experienced tool makers, to develop two new classes of genetically-encoded tools for trans-synaptic tracing. Both tools utilize designer proteins that are separately introduced (by viral infection) into potential ?sender? neurons and potential ?receiver? neurons in distinct regions of the mammalian brain. Receiver neurons that form direct synaptic contacts with sender neurons then activate a transcription factor or recombinase, which drives the expression of a reporter gene. For maximum versatility, the reporter gene can be a fluorescent protein to enable imaging, a channelrhodopsin to enable activity manipulation, or a tagged ribosome to enable omic analysis and assignment of neuronal subtype. Our plans include yeast-based directed evolution to engineer tool components, with the goal of maximizing signal-to-noise ratio for robust, specific, and sensitive tracing in vivo. Hence if we are successful, the outcome of this project should be two-fold: (1) a transformative toolkit of genetically-encoded reagents for trans-synaptic tracing in both anterograde and retrograde directions in the mammalian brain (that should be straightforwardly extensible to other model organisms such as fly, worm, and fish); and (2) discovery of new circuit elements in the mammalian brain that enable shared neurons to mediate distinct behaviors in the two sexes.